Summary: The SARS-CoV-2 pandemic (COVID-19) threatens the entire world?s health, economy and social stability and is likely to continue for the foreseeable future. The capacity of this virus to cause protean manifestations and resist public health control demonstrates its profound evolutionary and adaptive capacity. We have studied the experimental evolution and determinants of fidelity and adaptation of CoVs for more than 20 years. The parent grant (R01 AI108197) for this proposed supplement defines the determinants of CoV replicase proteins in virus fidelity and pathogenesis, and is specifically directed toward understanding the role of the unique CoV exoribonuclease encoded in nonstructural protein 14 (nsp14-ExoN). Using SARS-CoV, MERS- CoV and MHV, we have shown that nsp14-ExoN mediates RNA proofreading and is responsible for: i) CoV high fidelity replication; ii) resistance to nucleoside analog inhibitors; iii) virus fitness; iv) evasion of host immunity; and v) virulence in vivo. Engineered mutants of MHV and SARS-CoV lacking ExoN (ExoN(-)) are impaired in all of the above functions and thus define ExoN as an exceptionally conserved and vulnerable virus encoded target for inhibition and attenuation. In this administrative supplement, we propose in vitro and in vivo studies of SARS- CoV-2 nsp14-ExoN, with the long-term goal define its role in virus replication and as a target for inhibitors and attenuation. We will rescue SARS-CoV-2 mutants of nsp14-ExoN and define their impact on replication and disease. In Aim 1, we will introduce mutations into SARS-CoV-2CoV-2 nsp14-ExoN that are known in SARS- CoV, MERS-CoV, and/or MHV to abolish proofreading, alter nucleoside analog sensitivity, impact virus replication and fitness, or decrease virulence. Recovered viruses will be tested for these phenotypes. In Aim 2, we will select replication-competent ExoN mutants with defined phenotypes for testing in highly relevant human airway epithelial (HAE) cultures and in a mouse model for SARS-CoV-2 replication and disease. The long- standing and highly productive collaboration between the Denison and Baric labs has already resulted in development of SARS-CoV-2 reverse genetics, initial analysis SARS-CoV-2 recombination, and potential animal models, all of which, in combination with our established bioinformatics pipelines, will allow rapid progress on the proposed supplement aims and will provide data for longer-term detailed studies of the role of ExoN and the viral polymerase. The significance and urgency of these studies is high, as they will rapidly result in identification of nsp14-ExoN targets for small molecule inhibitors and multiple pathways to stable and universal attenuation of SARS-CoV-2 and future zoonotic CoVs.